Insulin action has been the subject of intense investigation for several decades. Interest in this field has been stimulated by an appreciation of the role of aberrant insulin signaling in several common metabolic disorders, including non-insulin-dependent diabetes mellitus and obesity. Although much progress has been made in understanding insulin signaling at the molecular level, many details remain unknown because of the unusual complexity of the effects of this hormone. Rather than corresponding to a simple series of linear pathways, insulin signaling can more accurately be characterized as a four-dimensional web of interacting molecules, involving coordinates in both space and time. Because of this complexity, it has been suggested that many important aspects of insulin signaling may only be unraveled via the use of cell-free systems that accurately reproduce various segments of the in vivo signaling cascades. The long-term goal of this proposal is to elucidate the molecular signaling cascades involved in the major metabolic effects of insulin in adipocytes. In order to facilitate the accomplishment of this goal, we have recently developed a novel cell-free system from 3T3L1 adipocytes to investigate insulin signaling. This system faithfully reconstitutes the PI 3-kinase signaling cascade from receptor autophosphorylation to events downstream of Akt activation, including the phosphorylation of GSK-3. We propose to further refine this cell-free system and to utilize it to pursue the following specific aims: 1) Akt is a serine kinase that plays a pivotal role in the PI3-kinase-dependent signaling pathway. It is activated by dual phosphorylations at a threonine and a serine residue. The threonine phoshorylation is catalyzed by PDK1, a well-characterized serine/threonine kinase, however, the serine kinase has not yet been identified. We have isolated a novel cytoskeletal subcellular fraction associated with adipocyte plasma membranes that is enriched approximately 1000-fold in PDK2 activity relative to total cellular homogenates. We propose to further enrich PDK2 activity from this fraction, identify the kinase using MALDI-TOF mass spectrometry, and then to clone and characterize the enzyme. 2) IRS-1 and IRS-2 are the major docking molecules that link the activated insulin receptor to the PI3-kinase signaling pathway. Current evidence suggests that IRS-1 and IRS-2 play distinct roles in the metabolic effects of insulin, but little is known about the mechanism by which these two IRS isoforms might mediate divergent signaling events. One hypothesis suggests that IRS-1 and IRS-2 promulgate distinct signaling outcomes by means of differential subcellular localization. Consistent with this hypothesis, we have observed that IRS-2, but not IRS-1, is highly enriched in the same plasma membrane-associated cytoskeletal fraction that contains the bulk of cellular PDK2 activity. Additionally, data obtained using our cell-free signaling assay indicate that Akt activation is dependent on plasma embrane-associated IRS-1, and that IRS-2 and cytosolic IRS-1 do not play a role in this process. Our second aim is to further characterize the novel PDK2/IRS-2-containing subcellular fraction and to determine the molecular basis and functional significance of IRS-2 targeting to the corresponding cellular compartment.